Defining signaling networks in epithelial homeostasis

ABSTRACT The goal of the work outline in this proposal is to understand the fundamental signaling that controls cell fate to maintain tissue homeostasis. Epithelial tissues demonstrate an intrinsic ability for their constituent cells to organize and maintain a steady-state of form and function. Many disease states lack these intrinsic controls. Epithelial tissues of the human body are in a constant state of renewal. Our understanding of the signaling systems that control how these complex epithelial tissues maintain robust organization is incomplete. Essential for progress, we need a quantitative understanding of signaling at the single-cell level in the context of physiological conditions to reveal systems-level behaviors that can be targeted therapeutically. We focus on protein kinases as critical mediators of signaling in the cell, which are well known to play prominent roles in tissue function and drivers of disease. Our kinome-wide studies have identified GSK3 and CLK3 as major tissue homeostasis regulators that govern the balance between proliferation and differentiation. In Project 1, we hypothesize GSK3 requires multiple suppressive inputs that uniquely produce different fate outcomes ranging from stem cells, transit-amplifying, and differentiated. We will provide the first systems-level mapping for multiple inputs onto GSK3 dynamics and how these dynamics are decoded into distinct cellular outcomes of renewing epithelium. In Project 2, we hypothesize CLK3 is a gatekeeper controlling stem cell fate through transcriptome regulation. We will define the activity of CLK3 in the stem cell niche as a regulator of expression and splicing of Wnt-target genes to promote stemness. Our approach uses high-throughput quantitative microscopy to measure single-cell behaviors in physiological organoid homeostatic culture models. Our research will define the regulatory mechanisms for two critical kinases, GSK3 and CLK3, and discover novel signaling circuitry needed for the accurate organization of renewing and regenerative epithelia, uncovering new strategies for treating diseases of regenerative tissues.